We are directing our research efforts towards expanding the current genetic tools applicable to the analysis of mammalian cells and their viruses. Specifically, we are attempting to isolate nonsense mutants and nonsense suppressors in cultured mammalian cells. The nonsense suppressor system would be valuable in the analysis of mammalian cells and their viruses because it should be a stringent conditional lethal system and because it will permit direct biochemical identification of the mutant gene products. For this purpose we have isolated 500 independent cell lines lacking hypoxanthine guanine phosphoribosyl transferase activity. These cell lines have been screened for immunological cross reacting material (CRM). About 40% of the HGPRT ion cell lines were CRM ion, as measured by a specific radioimmune precipitation assay. Some of these mutants have been identified as nonsense mutants. Revertants of these mutants are being assayed in vitro for suppressor tRNA. The in vitro system was derived from the parental L cells and is programmed with RNA from bacteriophages containing nonsense mutations or mammal messenger RNAs. The system responds to suppressor tRNA from E. coli and was used to prove that yeast super-suppressors contain nonsense suppressor tRNAs. The HGPRT mutants also afford an opportunity to study selective degradation of abnormal proteins and the processing of proteins in mammalian cells. These studies will be extended using both biochemical and genetic approaches.